| Assay Method Information | |
| | Binding Affinity Assay |
| Description: | SPA: Determination of equilibrium dissociation constant Kd of radioligand: Membrane prepared as described above was pre-incubated with PVT-WGA SPA beads (Perkinelmer Cat #RPNQ0003) at a ratio of 0.3 mg beads with 5 μg membrane per 25 μL binding buffer at 20° C. for 2 hours with gental shaking. This binding solution was centrifuged at 400 g for 5 minutes to collect the bead/membrane mixture. After re-suspending the pellet in binding buffer at the same calculated volume with 0.01% BSA (Sigma A1933), the bead/membrane mixture was added in 384-well low-binding surface plate (PerkinElmer Cat #6057480) at 25 μl/well. Radioligand at different concentrations with and without the non-radio-labeled same ligand 5 uM (for nonspecific and total signal, respectively) was added to bring final volume to 50 l/well with DMSO concentration at 0.1%. At equilibrium (3 hours after ligand addition), radiometric signal CPM was counted by using a Microbeta2 microplate counter (Perkinelmer). The Kd was determined by nonlinear regression fitting of specific signal plot against the concentration of radioligand [3H]-Ifenprodil. |
| Affinity data for this assay | |
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